Cells, tissues, organs, organisms and extracts thereof contain a variety of known and yet to be characterized proteases. Many of these proteases interfere with the expression, extraction and purification of desired proteins from natural sources or from recombinant organisms. Custom production of proteins for pharmaceutical research or medical applications can be compromised by the presence of proteases in extracts. Elimination of proteolysis during protein purification is difficult and further complicated by a lack of knowledge about the proteases present and the nature of the protease specifically cleaving the protein being purified.
Assays for detecting protease activity are known in the art. For example, fluorescence resonance energy transfer (FRET)-based methods are disclosed in U.S. patent application Ser. Nos. 10/477,044 and 10/343,977. These assays employ two fluorescent proteins attached via a protease cleavage linker, wherein cleavage of the linker results in a reduction in the level of FRET.
Moreover, U.S. Pat. No. 6,680,178 discloses a fluorogenic moiety, such as 7-amino-4-carbamoylmethyl-coumarin, covalently bound to a peptide sequence for identifying the primary and extended specificity of enzymes such as proteases.
Needed in the art is a rapid, cost-effective assay that can be used to quantitate protease activity, as well as be used to identify the type of protease being assayed. The present invention meets this long-felt need.